Determination Of Binding Constants Research Articles

A comprehensive study on synthesis and crystal structures of Cu(II) and Ni(II) complexes: In vitro and in silico evaluation of biomolecular interactions, antiproliferative activities and molecular docking

Cu(II) and Ni(II) complexes, [Cu(NNN)2](NO3)2 (1) and [Ni(NNN)2](NO3)2 (2) (NNN-donor ligand: 2,2′:6′,2″-terpyridine), have been synthesized and characterized by electronic absorption spectroscopy, CHN analysis, FTIR, ESI-MS and X-ray crystallography techniques. Interaction of the complexes 1 and 2 with biomolecules (calf thymus DNA (CT-DNA) and bovine serum albumin (BSA)) has been investigated by electronic absorption and fluorescence spectroscopy. The results show that the complexes 1 and 2 can bind to CT-DNA via a moderate intercalation mode. Moreover, the fluorescence quenching mechanism between the complexes 1 and 2 and BSA is a static quenching process. The antiproliferative activities of the complexes 1 and 2 against human breast cancer (MCF-7) and lung cancer (A549 and H1299) cell lines were investigated. The complex 1 was found to have promising antiproliferative activity in selected cell line, with lower IC50 values than cisplatin. Molecular docking studies conducted across the complexes 1 and 2 have provided their potential as prospective chemotherapeutic agents against tumors. The complexes 1 and 2 demonstrated spontaneous binding with targets including anaplastic lymphoma kinase (ALK), heat shock protein 90 (Hsp90), epidermal growth factor receptor (EGFR), bovine hemoglobin (BHb), human epidermal growth factor receptor 2 (HER2), and B-DNA. These interactions are primarily driven by van der Waals forces and π-π interactions. Determining binding constants and the multiplicity of binding sites enhances understanding of these molecular interactions.

ESI-MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I).

Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization-mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.

Erbium(III) complexes with fluoroquinolones: Structure and biological properties

Four erbium(III) complexes with the fluoroquinolones enrofloxacin, levofloxacin, flumequine and sparfloxacin as ligands were synthesized and characterized by a wide range of physicochemical and spectroscopic techniques as well as single–crystal X–ray crystallography. The compounds were evaluated for their activity against the bacterial strains Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Xanthomonas campestris, which was higher than that of the corresponding free quinolones. The interaction mode of the complexes with calf–thymus DNA is via intercalation, as suggested by diverse studies such as UV–vis spectroscopy, DNA–viscosity measurements and competitive studies with ethidium bromide. Fluorescence emission spectroscopy revealed the high affinity of the complexes for bovine and human serum albumin and the determined binding constants suggested a tight and reversible binding of the compounds with both albumins.

Constant-pH Simulations of a Coarse-Grained Model of Polyfunctional Weak Charged Biopolymers

A coarse-grained model of linear polyfunctional weak charged biopolymers was implemented, formed of different proportions of acid-base groups resembling the composition of humic substances. These substances are mainly present in dissolved organic matter in natural water. The influence of electrostatic interactions computing methods, factors concerning the structure of the chain, different functional groups, and the ionic strength on polyelectrolytes were studied. Langevin dynamics with constant pH simulations were performed using the ESPResSO package and the Python-based Molecule Builder for ESPResSo (pyMBE) library. The coverage was fitted to a polyfunctional Frumkin isotherm, with a mean-field interaction between charged beads. The composition of the chain affects the charge while ionic strength affects both the charge and the radius of gyration. Additionally, the parameters intrinsic to the polyelectrolyte model were well reproduced by fitting the polyfunctional Frumkin isotherm. In contrast, the non-intrinsic parameters depended on the ionic strength. The method developed and applied to a polyfunctional polypeptide model, that resembles a humic acid, will be very useful for characterizing biopolymers with several acid-base functional groups, where their structure, the composition of the different functional groups, and the determination of the main intrinsic proton binding constants and their proportion are not exactly known.

Delving into the Variability of Supramolecular Affinity: Self-Ion Pairing as a Central Player in Aqueous Host-Guest Chemistry.

The determination of binding constants is a key matter in evaluating the strength of host-guest interactions. However, the profound impact of self-ion pairing on this parameter is often underrated in aqueous solution, leading in some cases to a misinterpretation of the true potential of supramolecular assemblies. In the present study, we aim to shed further light on this critical factor by exploring the concentration-dependent behavior of a multicharged pillararene in water. Our observations reveal an extraordinary 1-million-fold variability in the affinity of this macrocycle toward a given anion, showcasing the highly dynamic character of electrostatic interactions. We argue that these findings bring to the forefront the inherent determinism that underlies the estimation of affinity constants, a factor profoundly shaped by both the sensitivity of the instrumental technique in use and the intricacies of the experimental design itself. In terms of applications, these results may provide the opportunity to optimize the operational concentrations of multicharged hosts in different scenarios, aiming to achieve their maximum efficiency based on the intended application. Unlocking the potential of this hidden variability may pave the way for the creation of novel molecular materials with advanced functionalities.

Delving into the Variability of Supramolecular Affinity: Self‐Ion Pairing as a Central Player in Aqueous Host‐Guest Chemistry

AbstractThe determination of binding constants is a key matter in evaluating the strength of host–guest interactions. However, the profound impact of self‐ion pairing on this parameter is often underrated in aqueous solution, leading in some cases to a misinterpretation of the true potential of supramolecular assemblies. In the present study, we aim to shed further light on this critical factor by exploring the concentration‐dependent behavior of a multicharged pillararene in water. Our observations reveal an extraordinary 1‐million‐fold variability in the affinity of this macrocycle toward a given anion, showcasing the highly dynamic character of electrostatic interactions. We argue that these findings bring to the forefront the inherent determinism that underlies the estimation of affinity constants, a factor profoundly shaped by both the sensitivity of the instrumental technique in use and the intricacies of the experimental design itself. In terms of applications, these results may provide the opportunity to optimize the operational concentrations of multicharged hosts in different scenarios, aiming to achieve their maximum efficiency based on the intended application. Unlocking the potential of this hidden variability may pave the way for the creation of novel molecular materials with advanced functionalities.

Application of singular value decomposition analysis: Insights into the complex mechanisms of amyloidogenesis

Amyloidogenesis, with its multifaceted nature spanning from peptide self-assembly to membrane-mediated structural transitions, presents a significant challenge for the interdisciplinary scientific community. Here, we emphasize on how Singular Value Decomposition (SVD) can be employed to reveal hidden patterns and dominant modes of interaction that govern the complex process of amyloidogenesis. We first utilize SVD analysis on Circular Dichroism (CD) spectral datasets to identify the intermediate structural species emerging during peptide-membrane interactions and to determine binding constants more precisely than conventional methods. We investigate the monomer loss kinetics associated with peptide self-assembly using Nuclear Magnetic Resonance (NMR) dataset and determine the global kinetic parameters through SVD. Furthermore, we explore the seeded growth of amyloid fibrils by analyzing a time-dependent NMR dataset, shedding light on the kinetic intricacies of this process. Our analysis uncovers two distinct states in the aggregation of Aβ40 and pinpoints key residues responsible for this seeded growth. To strengthen our findings and enhance their robustness, we validate those using simulated data, thereby highlighting the physical interpretations derived from SVD. Overall, SVD analysis offers a model-free, global kinetic perspective, enabling the selection of optimal kinetic models. This study not only contributes valuable insights into the dynamics but also highlights the versatility of SVD in unravelling complex processes of amyloidogenesis.

Synthesis and Characterization of Ruthenium-Paraphenylene-Cyclopentadienyl Full-Sandwich Complexes: Cytotoxic Activity against A549 Lung Cancer Cell Line and DNA Binding Properties.

Novel full-sandwich (η5-Cp)-Ru-paraphenylene complexes with the general formula [(η5-Cp)nRu(η6-L)](PF6)n where n = 1-3 and L = biphenyl, p-terphenyl and p-quaterphenyl, were synthesized and characterized by means of spectroscopic and analytical techniques. The structures of the complexes [(η5-Cp)Ru(η6-biphenyl)](PF6) (1), [(η5-Cp)Ru(η6-terphenyl)](PF6) (3) and [(η5-Cp)2Ru(η6-terphenyl)](PF6)2 (4) was determined by X-ray single crystal methods. The interaction of the complexes [(η5-Cp)Ru(η6-quaterphenyl)]Cl, (6)Cl, and [(η5-Cp)2Ru(η6-quaterphenyl)]Cl2, (7)Cl2, with the DNA duplex d(5'-CGCGAATTCGCG-3')2 was studied using NMR techniques. The results showed that both complexes interacted non-specifically with both the minor and major grooves of the helix. Specifically, (6)Cl exhibited partial binding through intercalation between the T7 and T8 bases of the sequence without disrupting the C-G and A-T hydrogen bonds. Fluorometric determination of the complexes' binding constants revealed a significant influence of the number of connected phenyl rings in the paraphenylene ligand (L) on the binding affinity of their complexes with the d(5'-CGCGAATTCGCG-3')2. The complexes (6)Cl and (7)Cl2 were found to be highly cytotoxic against the A549 lung cancer cell line, with complex (6) being more effective than (7) (IC50 for (6)Cl: 17.45 ± 2.1 μΜ, IC50 for (7)Cl2: 65.83 ± 1.8 μΜ) and with a selectivity index (SI) (SI for (6)Cl: 1.1 and SI for (7)Cl2: 4.8).

Direct Determination of High-Affinity Binding Constants by Continuous Injection Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a method to determine thermodynamic values (ΔG, ΔH, and ΔS) for ligand-receptor binding in biological and abiological systems. It is challenging to directly determine subnanomolar dissociation constants using a standard incremental injection approach ITC (IIA-ITC) measurement. We recently demonstrated a continuous injection approach ITC (CIA-ITC) [ J. Phys. Chem. B 2021, 125, 8075-8087]enables the estimation of thermodynamic parameters in situ. In this work, we demonstrate a label-free and surface modification-free CIA-ITC to determine the complete binding thermodynamics of a ligand with a subnanomolar dissociation constant KD. The KD for desthiobiotin (DTB)-avidin binding was determined to be 6.5 pM with respect to the ligand by CIA-ITC, a quantity unsuccessfully measured with IIA-ITC and surface plasmon resonance spectroscopy (SPR). This value compares well with literature-reported spectroscopic determination of DTB-avidin binding. Criteria with respect to the concentration of the ligand and receptor and flow rate for obtaining true equilibrium dissociation constants without displacement titration are presented.

Quercetin-phenylalanine 3d-transition metal-based {Co(II), Ni(II) & Cu(II)} intercalative therapeutic agents: DNA & BSA interaction studies in vitro and cleavage activity

New Quercetin-phenylalanine metal-based therapeutic agents of the formulation [Qu(Phe)M(II).(H2O)2].NO3 where M(II) = Co(II) and Ni(II) and [Qu(Phe)Cu(II).(H2O)2] were synthesized and their structure was predicted by IR, UV–vis, EPR and ESI-MS spectroscopic techniques. The bio-molecular interaction studies of the Quercetin-phenylalanine complexes, 1–3 with ct-DNA and BSA were performed using a battery of complimentary biophysical techniques. The corroborative results of these experiments revealed strong binding propensity via electrostatic interactions probably through minor grove binding towards ct-DNA, therapeutic target. The binding affinity of Quercetin-phenylalanine complexes 1–3 was quantified by determining binding constants values, Kb, Ksv, and the magnitude of binding propensity followed the order 3 > 1 > 2, implicating the preferential binding of Cu(II) complex 3 with ct-DNA. The cleavage studies were performed with complexes using gel electrophoretic mobility assay. The complexes 1–3 demonstrated efficient cleaving ability by the hydrolytic cleavage pathway involving hydroxyl (OH) radicals. BSA binding profile of Quercetin-phenylalanine metal therapeutics 1–3 was studied in order to understand the drug carrier potential of these compounds and found that complex 3 was capable of binding preferentially with BSA as compared to other complexes.

Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis

Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20–0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.

A Novel Flow Cytometry-Based Assay for the Identification of HCN4 CNBD Ligands.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are promising therapeutic targets because of their association with the genesis of several diseases. The identification of selective compounds that alter cAMP-induced ion channel modulation by binding to the cyclic nucleotide-binding domain (CNBD) will facilitate HCN channel-specific drug development. In this study, a fast and protein purification-free ligand-binding approach with a surface-displayed HCN4 C-Linker-CNBD on E. coli is presented. 8-Fluo-cAMP ligand binding was monitored by single-cell analysis via flow cytometry, and a Kd-value of 173 ± 46 nM was determined. The Kd value was confirmed by ligand depletion analysis and equilibrium state measurements. Applying increasing concentrations of cAMP led to a concentration-dependent decrease in fluorescence intensity, indicating a displacement of 8-Fluo-cAMP. A Ki-value of 8.5 ± 2 µM was determined. The linear relationship of IC50 values obtained for cAMP as a function of ligand concentration confirmed the competitive binding mode: IC50: 13 ± 2 µM/16 ± 3 µM/23 ± 1 µM/27 ± 1 µM for 50 nM/150 nM/250 nM/500 nM 8-Fluo-cAMP. A similar competitive mode of binding was confirmed for 7-CH-cAMP, and an IC50 value of 230 ± 41 nM and a Ki of 159 ± 29 nM were determined. Two established drugs were tested in the assay. Ivabradine, an approved HCN channel pore blocker and gabapentin, is known to bind to HCN4 channels in preference to other isoforms with an unknown mode of action. As expected, ivabradine had no impact on ligand binding. In addition, gabapentin had no influence on 8-Fluo-cAMP's binding to HCN4-CNBD. This is the first indication that gabapentin is not interacting with this part of the HCN4 channel. The ligand-binding assay as described can be used to determine binding constants for ligands such as cAMP and derivatives. It could also be applied for the identification of new ligands binding to the HCN4-CNBD.

Determination of dissociation constants via quantitative mass spectrometry

The interplay of biomolecules governs all cellular processes. Qualitative analysis of such interactions between biomolecules as well as the quantitative assessment of their binding affinities are essential for the understanding of biochemical mechanisms. As scientific interest therefore moves beyond pure structural investigation, methods that allow for the investigation of such interactions become increasingly relevant. In this perspective we outline classical methods that are applicable for the determination of binding constants and highlight specifically mass spectrometry based methods. The use of mass spectrometry to gain quantitative information about binding affinities however is a still developing field. Here, we discuss different approaches, which emerged over the last years to determine dissociation constants (KD) with mass spectrometry based methods. Specifically, we highlight the recent development of quantitative Laser Induced Liquid Bead Ion Desorption (qLILBID) mass spectrometry for the example of double stranded deoxyribonucleic acids as well as for different RNA—RNA binding protein systems. We show that quantitative laser induced liquid bead ion desorption can successfully be used for the top down investigation of complexes and their dissociation constants values ranging from low nM to low µM affinities.

Benzo[d]thiazole-2-carboxamides as new antituberculosis chemotypes inhibiting mycobacterial ATP phosphoribosyl transferase.

Aims: The screening of antimycobacterial benzo[d]thiazole-2-carboxamides against ATP-phosphoribosyl transferase (ATP-PRTase) was conducted. Materials & methods: The antitubercular potential of compounds1 and 2 against ATP-PRTase was assessed through the determination of half maximal effective concentration (EC50) andbinding constant (Kd), as well ascompetitive inhibitory studies and studies of perturbation of secondary structure, molecular modelingand L-histidine complementation assay. Results & conclusion:Compounds 1n and 2a significantly inhibited ATP-PRTaseas evidenced by their EC50 and Kdvaluesand the perturbation of thesecondary structure study. Compound 1n exhibitedstronger competitive inhibition toward ATP compared with 2a. The inhibition of the growth of Mycobacterium tuberculosis by targeting the L-histidine biosynthesis pathway and molecular modeling studies further supported theinhibition of ATP-PRTase.

Biocompatibility and biological activity of C70 fullerene adduct with L-threonine (C70(C4H9NO3)2)

The aim of this work is to synthesise and study the biocompatibility and biological activity of the C70 fullerene adduct with l-threonine (C70-Thr). The obtained adduct was identified using a complex of physicochemical methods, namely, 13C NMR spectroscopy, IR spectroscopy, thermogravimetric analysis, electron spectroscopy, elemental analysis, and high-performance liquid chromatography. The study of biocompatibility and biological activity of the C70-Thr adduct included the study of haemocompatibility (haemolysis, platelet aggregation, plasma coagulation haemostasis, binding to human serum albumin, esterase activity), antiradical activity, cytotoxicity, cell proliferation, and interaction with DNA (determination of the DNA binding constant and genotoxicity).

Study on the interaction of anions with A2B cobalt corroles in non-aqueous medium

A series of five cobalt corrole complexes Co[(p-NO2Ph)2RCor](py)2 where R is functionalized with different aromatic aldehydes at meso positions, was examined in acetonitrile solvent. Three of the five complexes changed color to green in 10[Formula: see text] M acetonitrile solution. Their potential to sense anions was investigated using the colorimetric and spectroscopic approach in 14 different tetrabutyl ammonium salts of F[Formula: see text], CN[Formula: see text], CH3COO[Formula: see text], NO[Formula: see text], AsO[Formula: see text], AsO[Formula: see text], Cl[Formula: see text], Br[Formula: see text], I[Formula: see text], ClO[Formula: see text], H2PO[Formula: see text], S[Formula: see text], HS[Formula: see text]and PF[Formula: see text]. Co-1, Co-2 and Co-4 shows selectivity towards F[Formula: see text], CN[Formula: see text]and CH3COO[Formula: see text] by their changing of color from green or reddish-brown to dark reddish-brown. The complexes exclusively bind these three anions at their axial positions. The binding constant determination gives the number of binding species (n) >1 at less than one equivalent concentration of anions in the acetonitrile solution of the complex. A band appeared at around 735 nm, on the other hand, suggests that a bis(CN)-derivative of corrole complex could form under >1 equivalent anionic conditions. The limit of detection for the Co-4 was superior than Co-1 and Co-2, which were 0.0068 [Formula: see text]M for CN[Formula: see text], 0.0082 [Formula: see text]M for F[Formula: see text]and 0.0177 [Formula: see text]M for CH3COO[Formula: see text] ion. Although the complexes detect all three anions at less than 1 equivalent of the anion addition, the Co-1 and Co-2 are more selective towards CH3COO[Formula: see text]ion, whereas Co-4 selectively detects CN[Formula: see text]anion at larger equivalents of these anions.

Label-Free Determination of the Kinetic Parameters of Protein-Aptamer Interaction by Surface Plasmon Resonance.

Due to their high specificity and affinity to target molecules, aptamers can be used as powerful tools in diagnostics, therapeutics, and environmental or food analytics. For the use in various applications, the detailed characterization of their binding behavior is an important step after selection to determine the interaction strength between the aptamer and its target and to find the best kinetics depending on the field of application. The surface plasmon resonance (SPR) spectroscopy is a powerful technology to investigate important parameters in molecular interaction, for example, kinetics, affinity, and specificity. The most-used system is the Biacore™ SPR system which comprises an optical biosensor for label-free monitoring of binding events in real time based on SPR. This biophysical phenomenon describes the changes in refractive index on a sensor surface which can be used to measure binding events and to determine kinetic constants. In this chapter, a detailed protocol for the determination of kinetic constants for protein-aptamer interaction is provided. An 82-nt long ssDNA aptamer which are targeted against human urokinase is used as a model system for determination of binding and dissociation constants using Biacore™ SPR technology. A detailed note section provides useful tips and pitfalls at the end of this chapter.

Evaluation of the effect of background electrolyte composition and independence of parameters in determining binding constants of betulin derivatives to β- and dimethyl-β-cyclodextrins by affinity capillary electrophoresis.

The values of the apparent binding constants for β-cyclodextrin complexes of betulin derivatives determined by mobility shift affinity capillary electrophoresis were found to be independent of the composition of the two background electrolytes used (tetraborate buffer, pH 9.18, and phosphate buffer, pH 8.00, both of them with 20 mM ionic strength). It has been found that if there is not a constant plateau on the binding curve then four independent parameters can be determined: binding constants (also referred to as stability, association, or formation constants) and ionic mobilities of 1:1 and 1:2 complexes. However, at least 10-12 data points in the binding curve should be used to reliably estimate the parameters. For the first time, the apparent binding constants for complexes of ester betulin derivatives with dimethyl-β-cyclodextrin have been determined by mobility shift affinity capillary electrophoresis. The logarithms of the constants for 1:1 and 1:2 complexes at 25°C for betulin 3,28-diphthalate with a 95% confidence interval are 4.98 (4.95-5.01) and 7.52 (7.26-7.68); for betulin 3,28-disulfate, the values are 4.97 (4.89-5.03) and 8.24 (6.82-8.52). It has been found that betulin 3,28-disuccinate forms only a 1:1 complex and the binding constant logarithm is 5.25 ± 0.02.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.